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(1)Methods in Cell and Molecular Biology

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(1)Methods in Cell and Molecular Biology. (chap 18: p727p727-734, 742742-744).. Reference book: Gerald Karp Cell and Molecular Biology: Concepts and Experiments( Experiments(5th edition) edition).

(2) The discovery of cells. 1665 - Robert Hooke (1635 (1635--1703) 1703)-- book Micrographia Micrographia,, published in 1665, devised the compound microscope, most famous microscopical observation was his study of thin slices of cork. Named the term “Cell “Cell”” - He wrote:.

(3) “. . . I could exceedingly plainly perceive it to be all perforated and porous. . . these pores, or cells cells,, . . . were indeed the first microscopical pores I ever saw, and perhaps, that were ever seen, for I had not met with any Writer or Person, that had made any mention of them before this.” Robert Hooke. Oxford University.

(4) Antoni van Leeuwenhoek (1665-1675), Dutch seller of clothes & buttons – in spare time, he ground lenses & made microscopes of remarkable quality A. He was the first to describe living single cells; his results were checked and confirmed by Hooke B. Saw “animalcules” in pond water (first to do this) using the scopes that he made.

(5) Overview of discovery.

(6) The Light Microscope: Principles. I. Components of the light microscope A. Light source - external to microscope or built into base (internal); illuminates specimen B. Condenser lens C. Objective lens – collects light rays focused on specimen by condenser lens D. Ocular lens.

(7) Objective lens: ~ collects 2 kinds of light rays passing through slide/specimen. 1. Rays altered by specimen – emanates from the many parts of the specimen; focused by objective lens in scope column forming real, enlarged image of the object 2. Rays not altered by specimen - pass directly into objective; form visual field background light.

(8) * Magnification = product of magnification produced individually by ocular & objective lenses * Resolution – the ability to see 2 neighboring points in a field as distinct entities ~ Resolution attained by a microscope is limited by diffraction. Empty magnification.

(9) •. Resolution is determined by the following equation & is limited by the wavelength of illumination & numerical aperture (N. A ). 1. d = minimum distance that 2 points in specimen can be resolved 2. λ = the wavelength of light (527 nm used for white light) 3. n = refractive index (RI) of medium present between the specimen & the objective lens 4. α = half the angle of cone of light entering objective lens 5. N. A., the denominator of the equation, is a measure of the light-gathering qualities of the lens (a constant for each lens).

(10) •. Resolution:. In air, air, largest possible angle of α = 90° 90°,  the maximum sin α = 1  n (RI of air) = 1.  N. A. = n sin α = 1 x 1 = 1. For an objective designed for use in air air,, largest N. A. is 1.0; 1.0; for an oil immersion objective objective,, the maximum possible N. A. is ~1.5.

(11) Rule of thumb (經驗法則): maximum useful magnification of a microscope = objective N. A. x 500 ~ 1,000 magnification beyond this amount, you get empty magnification & quality of image deteriorates Achieve high N. A. by using short focal length lens; allows lens placement very close to specimen Limit of resolution (LM): ~ 0.2 µm (200 nm) naked eye (N.A. = ~0.004): ~ 0.1 mm.

(12) Visibility ~ factors that allow an object actually to be observed; largely determined by contrast Ex : * clear glass bead placed in dark background  visible * clear glass bead placed in oil (with same refractive index as glass)  bead disappears. Contrast is the difference in appearance between adjacent parts of object or object & its background * ex: clearly visible stars in night sky.

(13) In microscope, you view light transmitted (or diffracted) through the object; to do this, object must be translucent (nearly transparent) However, … such objects are difficult to see So, to make thin, translucent specimens visible under scope  you must stain them with dye (dye should absorb only certain wavelengths within visible spectrum)  Those wavelengths not absorbed by dye are transmitted to eye, causing object to appear colored Ex. Feulgen staining. ~ specific for DNA (cannot be used with living cells). Bright--field microscope Bright. ~ suited for specimens of high contrast.

(14) Preparation of specimens for light microscopy I.. Two categories of specimens are observed in a light microscope – whole mounts & sections. II. Section production - kill cells by immersing in chemical solution called a fixative (formaldehyde, alcohol, acetic acid are the most common for the light microscope)  A good fixative rapidly penetrates cell membrane & immobilizes all of its macromolecular material, maintaining cell structure as close as possible to that of the living state  Tissue is then dehydrated by transfer through a series of alcohols & then embedded in paraffin (wax), which provides mechanical support during sectioning.

(15) Preparation of specimens for light microscopy (continue ~).  Slides containing adherent paraffin sections are immersed in toluene, which dissolves the wax.  The thin slice of tissue is thus left attached to slide where it can be stained or treated with enzymes, antibodies or other agents.  After staining, a coverslip is mounted over tissue using medium with same RI as slide & coverslip glass.

(16) The Light Microscope: Bright-Field and Interference Microscopy. Bright-field microscopy: ~ ideally suited for high contrast specimens (stained tissue sections); not good for all specimens.. Phase-contrast microscopy: ~ good for small, unstained specimens like living cells & those that are hard to see in bright-field. (type of interference microscopy; makes highly transparent objects more visible). Differential interference contrast (DIC) microscopes or Nomarski interference (after its developer): ~ another type of interference system.

(17) A ciliated protist. Bright--field Bright. Phase--contrast Phase. Differential interference contrast (DIC) (or Nomarskei)) optics Nomarskei.

(18) Phase--contrast microscopy: Phase A. Can distinguish different parts of object (organelles (organelles differ in refractive index (RI) since they differ in molecular composition)  turns differences in RI into differences in intensity (relative brightness & darkness) that are visible to the eye. B. Their ability to do this centers on ability of light waves to interact with one another (interference) 1. separates direct light waves (background) entering objective from diffracted light waves emanating from specimen 2. Causes light rays from the 2 sources to interfere with one another.

(19) Phase--contrast microscopy: (~ continue) Phase C. Useful in examination of intracellular components of living cells at relatively high resolution D. Disadvantages & limitations: ~ only suitable for observing single cells or thin cell layers 1. PP-C has optical handicaps that result in loss of resolution, and 2. The image suffers from interfering halos & shading produced where sharp changes in RI occur.

(20) Differential interference contrast (DIC) microscopes or Nomarski interference (after its developer): ~ another type of interference system ~ minimize the optical artifacts by completely separating direct & diffracted beams using complex light paths & prisms. A. Delivers an image that has an apparent 3-D quality B. Contrast in DIC microscopy depends on rate of change of RI across specimen; specimen; ~ edges of structures (where RI varies markedly over relatively small distance) are seen with especially good contrast.

(21) The Light Microscope: Fluorescence Microscopy. ~ Fluorescence microscopy is based on compounds called fluorochromes (fluorophores) that absorb invisible, UV light & release some of the energy as longer, visible light wavelengths. (fluorescence).

(22) Fluorescence Microscopy (~ continue) A. Presence of fluorochromes in cell is observed in fluorescence microscope fitted with UV light source; B. Fluorescence microscope light source produces a beam of UV light that is passed through a filter that blocks all wavelengths except the one that excites the fluorochrome C. The beam of monochromatic light is focused on the specimen containing fluorochrome, which becomes excited & emits light of visible wavelength that is focused by objective lens into an image D. Because the light source produces only UV (black) light, fluorochrome-stained objects appear brightly colored against a black background, providing very high contrast.

(23) Fluorescence Microscopy.

(24) Applications of fluorescence ~ use of fluorescence to localize molecules in the cell A. In one of its most common applications, one covalently links or conjugates a fluorochrome (fluorescein or rhodamine) to an Ab to make a fluorescent Ab;  Fluorescent Ab is used to determine location of a specific protein in the cell (immunofluorescence) B. Fluorochromes can also used in studies to locate DNA or RNA molecules that contain specific nucleotide sequences by using fluorescently labeled probes (ex. FISH).

(25) Immunofluorescence staining: ((免疫螢光染色 免疫螢光染色))  fix the cells (fixation)  permeabilize the cells  blocking Similar to Western blotting. Ab)  incubate with primary antibody (1o Ab)  incubate with secondary antibody (2o Ab) Ab)  mounting fluorescent Ab: Ab: ~ a fluorochrome (fluorescein or rhodamine)) is covalently linked rhodamine (conjugated) to an Ab to. Ag Ag. Ag Ag.

(26) Immunofluorescence staining • Direct immunofluorescence: – Ab ( specific to tissue Ag) is labeled with fluorochrome.. Fluorochrome Labeled Ab. FITC--conjugated Ab FITC FITC. Cell or Tissue Section as Ag.

(27) Immunofluorescence staining Indirect immunofluorescence immunofluorescence:: •. •. FITC conjugated goat antianti-mouse IgG. Ab specific to tissue Ag is unlabeled Fluorochrome-labeled antiFluorochromeanti-Ig is used to detect & binding of the first Ab.. • Qualitative to Semi Semi-Quantitative. Unlabeled Primary Ab. Fluorochrome Labeled AntiAnti-Ig. Cell or Tissue Section as Ag Primary Ab: α-β-tubulin (mouse).

(28) Fluorescent microscope 1. 2. 3. 4. filter. Fluorescin--conjugated 2oAb Fluorescin. 1oAb Cytoplasmic antigens Nuclear antigens. 3T3 or 3T3/ras mouse fibroblast. Glass slide.

(29) Immunofluorescence green -- tubulin red -- gamma tubulin blue -- DNA. red – actin blue -- DNA.

(30) Fluorescent Image of a Cell in Mitosis Spindle microtubules revealed with a green fluorescent antibody. Centromeres – red fluorescent antibody. DNA – blue fluorescent dye.

(31) Applications of fluorescence ~ use of fluorescently labeled proteins to study dynamic processes as they occur in a living cell (use of green fluorescent protein (GFP) from the jellyfish. Aequorea victoria) A specific fluorochrome can be linked to a cellular protein (e.g., actin, cytoplasmic dynein or tubulin) & the fluorescently labeled protein can then be injected into & followed through the living cell.

(32) Applications of fluorescence A noninvasive approach has been widely employed that utilizes a fluorescent protein (ex. GFP). Construct a recombinant DNA in which the GFPGFP-coding region is joined to the coding region of the protein being studied. transfect recombinant DNA into cells (synthesize a chimeric protein containing GFP fused to protein ). reveal dynamic activities in which the protein participates.

(33) EGFP EGFP--X fusion protein EGFP 35.

(34) Applications of fluorescence simultaneous usage of GFP variants  GFP variants that fluoresce in shades of blue (BFP), yellow (YFP) & cyan (CFP) have been generated through directed mutagenesis of the GFP gene  A distantly related red fluorescent protein (DsRed) has also been isolated from a sea anemone.

(35) Fluorescence Resonance Energy Transfer (FRET) FRET is based on fact that excitation energy can be transferred. from one fluorescent group (donor) to another fluorescent group (acceptor), as long as the 2 groups are very close together (1 – 10 nm).

(36) Fluorescence Resonance Energy Transfer (FRET)  measure changes in distance between two parts of a protein (or between two separate proteins within a larger structure).

(37) Confocal scanning light microscopy - replaces old serial section technology (slice embedded specimen); we now examine sections of specimen without cutting it with knife 1.. 2.. 3..

(38) http://micro.magnet.fsu.edu/primer/confocal/index.html.

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(40) LEICA TCS SP2 AOBS: AOBS: UV-VIS UVVIS--MP Dual monitors Scanner Fluorescence Microscope with HBO lamp. High NA Objectives. Laser power. Z-stage for Sectioning. Anti--vibration table Anti. Power switch For microscope. Control Panel. PC power Scanner power Fan power Visible lasers box & merge device.

(41) Why use cultured cells in research? ~ study cells & cell function in simplified, controlled, in vitro system; can remove cells from influences they are normally subject to within a complex multicellular organism 1. Cultured cells can be obtained in large quantity 2. Most cultures contain only a single cell type 3. Many different cellular activities can be studied in cell culture 4. Cells can differentiate in culture 5. Cultured cells respond to treatment with drugs, hormones, growth factors & other active substances.

(42) First vertebrate cell culture (1907)  Early culture media included a great variety unknown substances; even today, most media contain serum (lymph, blood serum, embryo homogenates)).  Cell culturists are trying to develop defined, serumfree media that support cell growth Composition of these chemically defined media is a relatively complex mix of nutrients, vitamins, a variety of purified proteins (insulin, epidermal growth factor, transferrin [provides cells with iron]).

(43) Since they are so rich in nutrients, tissue culture media invite the growth of microorganisms maintain sterile conditions within their working space use sterile gloves, sterilize all supplies & instruments, employ low antibiotic levels in media and conduct activities beneath a sterile hood. 器皿處理. 廢棄物處理. 一、純水、血清瓶或其他器皿:滅菌處理 二、培養基:過濾滅菌處理 (0.2 µm filter) 三、其他:70%酒精擦拭 培養基:滅菌或漂白水處理 其他:滅菌處理.

(44) Process of cell culture: secondary culture. ~ get cells (often previously cultured cells frozen in liquid N2), thaw them & place them in medium; called secondary culture since cells are derived from a previous culture Process of cell culture: primary culture. ~ get cells directly from organism, most animal cell primary cultures are from embryos. 1. Take tissue from embryos & treat with proteolytic enzyme, like trypsin 2. Wash tissue free of enzyme & usually suspend cells in saline solution lacking Ca2+ ions & containing a substance like ethylenediamine tetraacetate (EDTA).

(45) Type of primary cell culture – mass & clonal culture Mass culture ~ a relatively large number of cells is added to culture dish. - Cells settle & attach to dish bottom & form relatively uniform cell layer; surviving cells grow & divide ( monolayer monolayer)). Clonal culture ~ a relatively small number of cells is added to a dish so that each cell resides at some distance from its neighbors.

(46) Normal (nonmalignant) cells can only divide a limited number of times (~50-100) before they senesce & die  many cells commonly used in tissue culture studies have undergone genetic modifications * grow indefinitely (such cells are called a cell line) * can grow into malignant tumors, if injected into susceptible laboratory animals. transformation. Human cell lines (e.g., HeLa cells) are typically derived from human tumors or from cells treated with cancercancercausing viruses or chemicals.

(47) 細胞庫簡介 一、ATCC、BCRC介紹 ATCC (American Type Culture Collection ): http://www.atcc.org/ BCRC (Bioresource Collection and Research Center, 生物資源保存及研究中心): http://www.bcrc.firdi.org.tw/wwwbcrc/main.jsp. 二、細胞庫資料判讀介紹.

(48) http://www.atcc.org/.

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(54) Microbiological Safety Cabinets Class I - air is drawn from the room through the open front, and over the working area. It is then passed through high efficiency particulate air (HEPA) filters which remove infectious particles, and is ducted to outside air. A minimum airflow of 0.7 m/s must be maintained through the front of the cabinet. Filters must be changed when the airflow falls below this level. 保護工作者優先,提供無菌環境次之.

(55) Microbiological Safety Cabinets Class II - air is filtered and most of it is recirculated through the cabinet. This cabinet protects the work as well as the worker. About 70% of the air is recirculated through filters so that the working area is bathed in clean (almost sterile) air. The remaining 30% of air is exhausted to the atmosphere and is replaced by a "curtain" of room air which enters at the working face. 提供無菌環境與保護工作者 兼顧,適合細胞培養.

(56) Microbiological Safety Cabinets Class III - Class III cabinets are totally enclosed and leakproof. The operator works with gloves which are sealed into the front of the cabinet by removable gaskets. 密閉空間,用於高度 感染性操作.

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